A variety of stimuli have been reported to induce apoptosis in endothelial cells. Unlike other cell types in which considerable information exists regarding intracellular survival signals, anti- apoptotic signals for endothelial cells have been described only in the extracellular space including basement membrane proteins, integrins and attachment per se. Alternatively, significant evidence exists to support a role for reactive oxygen intermediates as signaling molecules in the apoptotic pathway and thus, we suggest that intracellular antioxidants may interfere with apoptosis in endothelial cells. Accordingly I will test this hypothesis in a simple model system of E. coli. [lipopolysaccharide (LPS)]-induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC) that my sponsor recently described. I will initially characterize the early sequence events that occur after exposure of wild type SPAEC to LPS including: a) lucigenin-derived chemiluminescence detection of superoxide anion; and b) changes in gene expression of p53, Bcl-2 and Bax (Northern and Western blot). I will then genetically engineer SPAEC to overexpress either human CuZnSOD (via adenoviral vector) or metallothionein lla (via cationic liposome-mediated gene transfer) and repeat the above aims. In addition, apoptosis will be assessed biochemically (terminal transferase assay: internucleosomal DNA fragmentation) and morphologically (electron microscopy) 24 hours after exposure of SPAEC to LPS. These data should provide fundamental insight into the intracellular signaling role of reactive oxygen intermediates in LPS-induced apoptosis in pulmonary endothelium and suggest therapeutic strategies aimed at affecting the course of endothelial cell damage in acute lung injury.